Agrawal R , Tran U , Wessely O .

#5R21DK077763-03 NIDDK NIH HHS , R21 DK077763-02 NIDDK NIH HHS , R01 DK080745 NIDDK NIH HHS , R21 DK077763 NIDDK NIH HHS

             miRNA binding

	Fig. 1. Inhibition of miRNA biogenesis results in pronephric abnormalities. (A-E′) Xenopus embryos were injected with 6.4 pmol Dicer-MO or 1.07 pmol Dgcr8-MO and compared with uninjected sibling embryos by morphology at stage 43 (A-A′), histology with Hematoxylin and Eosin at stage 42 (B-B′), immunostaining with 3G8 and 4A6 at stage 40 (C-D′), and by whole-mount in situ hybridization for β1-Na/K-ATPase at stage 39 (E-E′). Arrowheads indicate edema formation (A′,A′) and the loss of 4A6 staining in duct (D′,D′). en, endoderm; no, notochord; nt, neural tube; pn, pronephros; s, somites. (F) Quantification of 4A6 staining in the pronephric duct, comparing uninjected control embryos with embryos injected with a standard control MO (Std-MO), Dicer-MO, Dgcr8-MO, a combination of Dicer-MO and Dgcr8-MO, or Dgcr8-MO together with Dgcr8* mRNA at stage 40. The graph represents the summary of at least three independent experiments. The number (N) of embryos analyzed is indicated above the bars. Black, normal expression; gray, partial expression; white, no expression. (G) RT-PCR analysis of multiple miRNAs after Dicer and Dgcr8 knockdown at stage 35. SNO-412 served as loading control.
	Fig. 2. Inhibition of miRNA biogenesis affects patterning of the pronephros. (A-G′) Whole-mount in situ hybridization for markers of terminal pronephros differentiation on uninjected and Dgcr8-MO-injected Xenopus embryos at stage 39. (A,A′) SGLT1-K; (B,B′) NKCC2; (C,C′) NBC1; (D,D′) ClC-K; (E,E′) Cadherin-16; (F,F′) ROMK; (G,G′) NCC. Arrowheads indicate a reduction in the intermediate and distal tubular domain. (H,I) Schematics illustrating the different pronephric regions and their corresponding marker gene expression (H) and the phenotype of Dicer-MO- or Dgcr8-MO-injected embryos showing a reduction in the proximal and intermediate tubules as well as in distal tubule DT1 (I).
	Fig. 4. miR-30a-5p knockdown and global inhibition of miRNA biogenesis have very similar kidney phenotypes. (A) To determine the specificity of miR-30a5p-MO, three consecutive miR-30a-5p binding sites (BS) were introduced in the 3′UTR of a lacZ reporter. Xenopus embryos were injected with the reporter in the presence or absence of miR-30a-5p duplex and miR-30a5p-MO and processed for lacZ staining at gastrula stage. Injections with miR-34b-MO served as a specificity control. (A′) Quantification of the lacZ staining in embryos: white, no staining; gray, partial staining; black, strong staining. The number of embryos analyzed in three different experiments is indicated above the bars. (B-H′) Xenopus embryos injected with miR-30a5p-MO and uninjected controls were analyzed by morphology (B,B′), histology (C,C′), immunohistochemistry with 3G8 and 4A6 (D-E′), and whole-mount in situ hybridization for Cadherin-16 (Cad-16; F,F′), ClC-K (G,G′) and NKCC2 (H,H′). Arrowheads indicate the presence of edema (B′), loss of 4A6 staining in the pronephric duct (E′), and the shortening of the tubule segments IT1, IT2 and DT1 (F′,G′,H′). en, endoderm; no, notochord; nt, neural tube; pn, pronephros; s, somites.
	Fig. 5. Apoptosis and proliferation do not contribute to the kidney phenotype. (A,A′) TUNEL staining of transverse sections from uninjected and miR-30a5p-MO-injected Xenopus embryos at stage 39. TUNEL-positive cells appear brown and are indicated by arrows. Note that no significant differences were observed by analyzing many serial sections of the pronephros. Several embryos and multiple independent experiments were analyzed. (B-B′) Immunofluorescence analysis of proliferation with an anti-phospho-Histone H3 antibody comparing transverse sections of uninjected control embryos, embryos injected with miR-30a5p-MO and embryos treated with aphidicolin and hydroxyurea (APC+HU) at stage 40. (C) Quantification of phospho-Histone H3-positive cells in the pronephros of uninjected (dark gray) and miR-30a5p-MO-injected (light gray) embryos at stages 37 and 40. Consecutive sections covering the entire pronephric tubular area were counted. Numbers are the average of at least four different embryos from two independent experiments. Error bars represent s.d. (D-F′) Immunostaining and in situ hybridization of untreated and APC+HU-treated embryos with 4A6 (D,D′), Cadherin-16 (Cad-16; E,E′) and NKCC2 (F,F′). Note the absence of any patterning differences, even though proliferation was completely inhibited.
	Fig. 6. Xlim1 is regulated by miR-30a-5p. (A-D′) Whole-mount in situ hybridization for Pax2 (A,A′), Pax8 (B,B′), Hnf1-β (C,C′) and Xlim1 (D,D′), comparing uninjected and miR-30a5p-MO-injected Xenopus embryos at stage 39. (E-G′) Whole-mount in situ hybridization for Xlim1 mRNA of uninjected and miR-30a5p-MO-injected Xenopus embryos at stages 31, 35 and 39. Note that Xlim1 expression is maintained at a higher level in miR-30a5p-MO-injected embryos after stage 35, when endogenous expression of miR-30a-5p can first be detected (arrows in D′,G′). (H,H′) Immunohistochemistry with 4A6 of stage 40 embryos injected with 4 pg pCS2-Lhx1* DNA into one blastomere at the 4-cell stage, comparing the left with the right side. Arrows indicate changes in 4A6 staining. Ectopic expression of Lhx1 mRNA transcribed from the pCS2-Lhx1* was confirmed by in situ hybridization (data not shown).
	slc12a3 (solute carrier family 12 member 3) gene expression in Xenopus laevis embryos, NF stage 39, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	atp1b1 (ATPase, Na+/K+ transporting, beta 1 polypeptide ) gene expression in a Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.

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